Oral cancer survival has not improved for the past 20 yrs. Recently, cancer stem cell (CSC)- based therapies have shown their potential to control cancer recurrence. However, the lack of in vivo models has hindered attempts to identify markers of oral CSC (OCSC) in their natural niche. This proposal will focus on identification of cell surface markers of normal oral stem cells (OSC) and OCSC allowing future therapeutic approaches to specifically target OCSC without affecting normal OSC. We will use two unique and complementary in vivo models for the proposed studies. The first model is a direct patient tumor xenograft model (DPTXM) in which individual patient tumors are transplanted into immune compromised mice and then passaged through several generations. The second model is a genetically engineered mouse model (GEMM) of oral cancer. Our unpublished data have shown that Smad4 single gene deletion (Smad4-/-) in oral epithelia results in oral cancer formation. Aim 1 will identify differences in cell surface markers between normal oral stem cells and OCSC. The Hoechst dye effluxed "side population" (SP) cells, which have been shown to be a common property of normal and cancer stem cells, will be used to sort cells from normal oral mucosa, preneoplastic and neoplastic tissues from the above two models. The SP cells and non-SP cells from these tissue samples will be subjected to a high throughput biotin/proteomics analysis for their cell surface proteins. Aim 2 will validate OCSC markers. Potential OCSC markers identified from Aim 1 will be used to sort oral tumor cells and test for their cancer initiation properties. To determine if non-SP tumor cells can behave like OCSC by acquiring the ability to self renew, we will use CSC markers identified from other cancer types to sort oral tumor cells and then assay for their tumorigenecity. Aim 3 will determine if surrounding fibroblasts affect OCSC properties and niche. First, we will examine the ratio of human oral cancer-associated fibroblasts (CAFs) vs. host mouse fibroblasts through serial passages of a transplant by in situ hybridization using probes specific for human and mouse chromosomes. OCSC markers and tumor initiation ability will be analyzed to determine if reduced CAFs affect OCSC markers and behavior. Second, we will transplant sorted OCSC positive cells with either wildtype fibroblasts or oncogenic fibroblasts harboring a deletion of the type II receptor of transforming growth factor [unreadable] (TGF[unreadable]RII- /-). We will examine if TGF[unreadable]RII-/- fibroblasts cause changes in OCSC markers and their tumor initiation properties. PUBLIC HEALTH RELEVANCE: This proposal will focus on identification of cell surface markers of oral stem cells and oral cancer stem cells allowing future therapeutic approaches to specifically target oral cancer stem cells without affecting normal oral stem cells.